I. Introduction
The role of biodegradation as a process technology for use in decontamination of hazardous pollutants is clearly accelerating. New uses which incorporate use of microbial populations are being found every day. The strong interest in biotechnology processes for waste cleanups stems from a combination of economic, national health and other environmental driving forces. It is evident that congressional limits on financial resources dictate that the most cost-effective processes be employed to ameliorate contaminated sediments and soils which threaten population health or key environmental habitats. For example, the movement of hazardous wastes through surface and subsurface soils if not alleviated could result in significant poisoning of potable water aquifers and discharge of hazardous contaminants into navigable streams and rivers and result in contamination of edible fish and shellfish which are native to these waters. The estimates to decontaminate soils containing hazardous mixed wastes are in the range of many billions of dollars when using current approved technologies. These technologies focus on processes which include thermal destruction, solidification and encapsulation. The current philosophy of the United States Environmental Protection Agency is to stall current high cost remediation of many hazardous waste sites until lower cost alternatives become available through development of new emerging technologies. There is a trend which favors developing natural insitu versus exsitu processes. The natural insitu processes are expected to have minimal construction and mobilization costs compared to large costs of alternatives for excavating or mixing contaminated soils for use in other natural insitu or exsitu technologies. The costs of remediation may be increased further when using disturbed soil in exsitu technologies by inadvertent contamination of uncontaminated soils. The natural insitu process of biodegradation is predicted to extend the time of remediation but is predicted to be the process of choice for remediation of carbon-based hazardous wastes when compared to the majority of competing remediation technologies.
II. Background
A. Hazardous Wastes and Soil Classification Systems.
1. HAZARDOUS WASTES.
Severe problems exist in removal and destruction of hazardous wastes both in impoundments and as subsurface hazardous contaminants. These subsurface contaminants are typically classified into three categories. These categories include radionuclides, heavy metals, and organic pollutants. Many subgroups exist in each category. Contaminants may exist individually or in combined groupings. The toxic pollutants pose a health threat to man through the potential for and possibility of leaching of these contaminants through fissures and/or permeable soils into aquifers or recharge areas which provide sources of potable water. The majority of hazardous wastes are contained in some form of impoundment which can be composed of a simple clay pit bulldozed out of the ground or as elaborate as a hazardous waste landfill where multiple man made and natural compacted clay layers are used to prevent any hazardous leachates from escaping from the enclosed landfill.
2. SOIL CLASSIFICATION SYSTEMS.
Soils are typically classified by three systems which include: geologic origin, agricultural origin and grain size of the material, and engineering classification systems which comprise a common classification based on grain size, American Association of State Highway and Transportation Officials (AASHTO) for suitability of material for highway construction, and the Unified Soil Classification System (USCS) which is based on general engineering behavior of the soil. The latter system uses grain-size distribution and the Atterberg limits to classify fine-grained soils. The subsurface soils are composed of a broad range of materials designated by particle size into sands, silts, and clays. The reactivity of a soil is based on the surface area of the particular soil. The highest reactivities are exhibited by those clays which have the highest surface areas. The reactivity or activity of a clay soil is defined by equation 1: ##EQU1## where A.sub.C is the activity of a clay and measures the colloidal behavior.
I.sub.P is the plasticity index and represents the range of water content where the clay is in the plastic state. PA1 Q.sub.h is the flow under hydraulic conditions in cm.sup.3 /sec. PA1 K.sub.h is the hydraulic conductivity or permeability in cm/sec. PA1 i.sub.h is the hydraulic gradient. PA1 A is the cross-sectional area in cm.sup.2. PA1 K.sub.e is the electro-osmotic coefficient of permeability in cm/sec)//(Volts/cm. PA1 i.sub.e is the electrical gradient in volts/cm. PA1 A is the cross-sectional area in cm.sup.2.
W.sub.L and W.sub.P are the Atterberg limits and define the plastic state for a clay.
%&lt;2 microns represents the percentage of clay in a soil.
Clays are called cohesive soils and in natural settings are characterized by low permeabilities and restrict the flow of fluids as well as the movement of microbial populations.
The primary properties of cohesive soils (clays), are summarized in Table 1. The important properties of clay with respect to biological activity within clay soils is that clays typically contain substantial quantities of water ranging from 10 percent to fully saturated. Moisture is necessary for the survival of microorganisms. Clay soils can be thought of as natural resin exchange beds. Clay soils exhibit specific cation exchange capacities, negative polarity, and adsorb positive cations from the pore fluid or groundwater that is slowly moving through the clay soil. The pores in the clay soils can be smaller than the diameter of the rod-shaped soil microorganisms and thus can restrict movement of the microbial populations in clay soils. Sandy soils, on the other hand, are characterized by low surface area and high permeability, and thus do not retain significant quantities of moisture. Sands and gravels are readily drained and constitute the major unsaturated vadose zones in subsurface soils. Organic pollutants and soluble hazardous metals will be partly entrapped in sandy soils while rainfall percolation will carry a fraction of these pollutants to deeper depths where the contaminants will either be trapped in low permeability clay soils or leach into flowing streams and rivers.
B. Natural Microorganisms in Soils.
3. SOIL SURFACE HABITAT AND SIZE.
Soil microorganisms tend to congregate at the soil surface in a shallow layer of approximately 10 centimeters in depth. This shallow layer is referenced as either the weathering layer or the plough layer. The large majority of food (leaf fall, plant and animal detritus, etc.) is available at the soil surface. Natural biodegradation end products are fulvic and humic acids which may take up to 25-30 years to biodegrade. Microbial population size bears a direct relationship to the availability of food sources. Table 2 shows a distribution of microorganisms in the initial 75 centimeters of a soil profile and includes aerobic bacteria, anaerobic bacteria, actinomycetes, fungi and algae. The total aerobic and anaerobic bacteria in the upper 8 cm. of soil was 77-80 percent of the total bacteria found in the 75 cm. profile. 95 percent of all bacteria were found in the upper 25 cm. of the soil profile. Aerobic bacteria averaged between 80-90 percent of the total bacteria for the soil horizons investigated.
The indigenous microbial population was measured in a semitropical location where insitu bioventing was in progress at a diesel fuel site. All measurements were taken at depths of 200- 280 cm. The soil contained only sandy and silty constituents and was very permeable. Standard plate counts reported as colony-forming units/gram varied from 10.sup.2 to 10.sup.5 CFU/gram.
Variances in the microbial population size show that the background microbial number (no diesel fuel) was between 10.sup.2 -10.sup.3 CFU/gram and a second background location containing diesel fuel was also in the same range. The microbial number range increased by a factor of 10 for the majority of the tests reported for the bioventing area or 10.sup.4 CFU/gram. The diesel fuel concentration varied from essentially zero (background) to a high of 7700 mg/kg. The highest microbial number 10.sup.5 CFU/gram was reported at the 200 cm. depth with a minuscule diesel fuel concentration of 0.2 mg/kg. It appears that increasing fuel concentrations inhibited the rates of microbial growth. Marks found analogous data for tests performed in high intensity continuous stirred aerobic sealed reactors when destroying API separator sludges containing step-up concentrations of highly toxic polynuclear aromatic (PNA) benzo(a)pyrene. An efficient process reactor would retain a microbial density between 10.sup.7 CFU/ml to 10.sup.9 CFU/ml. This density was maintained in long term study reactors for over 2 years using a natural adapted diverse microbial population. Marks found that, at 2,000-4,000 mg of benzo(a)pyrene per kg of dry feed solids, the microbial density varied from 10.sup.7 to 10.sup.8 CFU/ml. The microbial density declined to 10.sup.6 CFU/ml for all concentrations of benzo(a)pyrene tested between 10,000 up to 35,000 mg BaP/kg. These data also demonstrate the ability of an adapted natural microbial population to survive on increased dosing of a hazardous substrate. A one magnitude decline in population density occurred due to the inhibitory effect resulting from the increased dosing of the hazardous PNA. Nematodes and protozoa were observed to have difficulty surviving at the higher concentrations.
Bacteria have the capability of enlarging and armor plating the cell capsules by secretion of sticky materials for attraction of clay platelets or growing fibrils which trap clay particles. Electron microphotographs have shown rod bacteria growing fibrillar capsules and exuding a sticky film on the outside of the cell capsule for entrapment of clay platelets. Electron microphotographs of soil bacteria show that the bacteria can enlarge their diameters from 0.5-1.0 micrometers up to 1.0-1.5 micrometers through the armor plating techniques. The armor plated microbe has increased resistance to washout from the surface into deeper soil horizons where there is virtually no food supply and the armored bacteria also have better protection from predator attack. The majority of microbes strongly prefer to remain in the weathering layer of soils and use the mechanisms of natural cell enlargement and/or adhesion to large particles of soil to prevent wash-out.
4. RELATIVE SIZE OF SOIL CONSTITUENTS.
Table 3 shows the relative sizes of constituents in soils. It is noted that sand grains are very large (50-2,000 micrometers in diameter) and readily permit egress of the microbial populations through the large void spaces between the grains.
Contaminants are readily washed into sandy soils by natural rainfall and runoff. All varieties of microorganisms can readily move through the voids in sandy soils. Fungi and protozoa (14-600 micrometers in diameter) are found deeper in many sandy soils when sufficient moisture is present. Significant distribution of fungi in grass sod has been reported at 64-84 centimeters in depths.
Clay soil particles are very small (&lt;2 micrometers) and the interstitial and pore spaces in clays is typically from 0.3-10 micrometers in size. The majority of microorganisms are not able to effectively pass through clays due to pore size restrictions. Thus microbial populations in clay soils can be characterized by very low population density and small diameters. The permeabilities of stiff clays will vary from 10.sup.-6 cm/sec to 10.sup.-10 cm/sec. Natural groundwater or pore fluid flows are highly restricted by stiff clay horizons. Hazardous organic and soluble heavy metal contaminants will percolate through sand lenses and are often retained by clay horizons. Dense non-aqueous phase liquids (DNAPLs) are an example where the heavy hydrocarbons will sink downwards in a permeable aquifer and accumulate as a glob on top of the accumulations of silts and clays in the bottom of the aquifer.
C. Hazardous Organic Compounds and Heavy Metals in Soils.
1. ORGANIC POLLUTANTS FOUND IN SOILS AND DEGRADATION RATES.
The two general classifications include naturally-occurring organic compounds and anthropogenic organic compounds. The rise in new types of the second category has resulted in accumulations of man-made compounds in soils.
Generally the more complex organic structures and degree of halogenation will increase the bioresistance. These compounds may bio-accumulate in the environment if left to degrade in the natural surroundings. The slowest degradation rates are encountered when the organic pollutants undergo land treatment or composting. The organic compounds will degrade much faster in well mixed soil systems. Biodegradation rates have been listed for over 300 organic chemicals in insitu and disturbed soil environments. Some of the environments include: soil incubation, soil percolation, activated sludge, groundwater testing, natural acclimated microbial flora, continuous flow and static culture biodegradation flask. Biodegradation rates are expressed in terms of biological half-lives and are usually first order rates. The well mixed systems using acclimated microorganisms are often orders of magnitude faster but costs are much higher.
2. EFFECT OF HEAVY METALS ON SOIL MICROORGANISM PROCESSES.
The impact of heavy metals on biological activity appears to be specific to the biological process and metal involved. Hazardous metals such as Ag, Cd, Co, Cu, Hg, Ni, Pb, Sn, and Zn have been reported to inhibit respiration as the concentration of the toxic metal is increased. The Ph range for 36 tests varied between 4.8 to 6.75 with the average Ph at 5. This indicated that an acidic pH environment is preferred. The addition of an organic food source negated the impact of most metals. All test media was sandy loam, loamy sand or silt loam, and results in clay soils may be different.
It has been indicated that microorganisms have adapted mechanisms for surviving in soils with relatively high metal concentrations. Findings have indicated that: (1) some microorganisms have energy-driven efflux pumps that keep intercellular concentration of metals low by pumping the metal out of a cell, (2) some microorganisms can convert enzymatically and intercellularly a more toxic form of an element or metal into a less toxic form, (3) other microorganisms can synthesize intercellular polymers that trap and remove metals from the intercellular solution, (4) certain microorganisms can bind large amounts of metal ions to their cell surfaces via precipitation or by covalent or ionic binding and (5) some microorganisms can biomethylate metals and the methylated form can then be transported out of the cell by a diffusion-controlled process.
D. Selection of the Microbial Population for Degrading the Specific Waste.
3. ENGINEERED MICROBES.
Experience in observing field tests of genetically-altered or engineered microbes has not shown very positive results. Tagged microorganisms that are constructed in an aseptic laboratory environment seem to have a limited opportunity when placed in a hostile field environment. There is a scarcity of food in soils, and the natural indigenous microorganisms have learned to survive over a few million years. Genetically-altered microorganisms released into soils are another food source for hungry indigenous microbial populations. The engineered microorganism is constructed to perform a specific function such as nitrogen fixation on soybean roots. The engineered microbe must be placed near to the soybean plant so it can participate in infection and the symbiotic acquisition of N.sub.2. If the engineered microbe is not placed near a soybean root, then no infection will occur. The number and size of nodules which are related to infection by the engineered microorganisms must be compared to nodulation by the cross-inoculation group Rizobium Japonicum. Data has shown that at best nodulation by the altered microorganism was sometimes as efficient as the cross-inoculation group for symbiotic nitrogen fixation. There are also risk factors associated with use of an engineered organism.
4. ADAPTED AND ACCLIMATED INDIGENOUS MICROBIAL CULTURES.
An adapted microbial culture relates to the enhanced ability of the culture to survive and reproduce in a particular environment by natural modifications of the characteristics of the organisms. The environment in this instance is the organic contaminant in the insitu site. Acclimation is the lagtime period where indigenous cultures acquire the ability to degrade new and difficult organic pollutants. Both of these attributes must be acquired by an indigenous population to perform successful biodegradations.
One of the best methods found to develop a robust adapted microbial culture is to collect an indigenous population from contaminated media in the natural environment which is known to contain the hazardous waste of interest. This culture is composed of a multitude of species and already contains the genetic material necessary to degrade the waste. A stock culture can be maintained using a carbon source which contains the hazardous waste. For example, if one is planning an experiment to biodegrade high concentrations (100 mg/1) of benzo(a)pyrene, then one would look for a natural source of polynuclear aromatics (PNA's) that has been exposed to natural microorganisms. Excellent sources of microorganisms are contaminated soils adjacent to creosote-type wood preservation plants, and discarded creosote-dipped wood railroad ties or power poles. Chipped wood or sawdust also provides an extra benefit in that it is degradable (provides metabolic energy) and microorganisms can use this material for reaction sites. A stock culture from shredded railroad ties contained many types of bacteria including Pseudomonas and Athrobacter, actinomycetes, three varieties of fungi, five types of protozoa and nematodes. The adapted culture along with other biological and nutrients agents can be added to subsurface contaminated soils by mixing into extracted groundwater and re-injection of the biological agents-groundwater mixture.
One method to confirm that the developed culture is robust is to plate out the microbial culture in the laboratory using similar concentrations that were found in the contaminated media. Either pour plates or spread plates containing the appropriate concentrations of target toxic hydrocarbons should be plated on plain agar with the toxic waste as the only carbon source. Table 5 shows the pour plating of the inoculum of two adapted stock cultures using refinery API separator sludge and a petrochemical plant API separator sludge. Ten hazardous organic compounds were used to demonstrate that the cultures had developed the genetic responses for successful culture plating of 90 percent of the compounds. Plate counts are shown as culture-forming units/milliliter of stock culture (CFU/ml). Pour plating is preferred for measurement of microbial populations in subsurface soils, because microaerophilic organisms will grow on pour plates and not on spread plates. The enumeration of protozoa and nematodes is easily obtained by direct counting using a microscope. It should be recognized that all soil cultures are facultative and able to grow in oxygen-rich and oxygen-lean regimes. The oxygen-rich regimes are of course preferred as biodegradation rates will be approximately 100 times faster and result in a more cost-effective process.
5. MACRONUTRIENTS AND MICRONUTRIENTS.
The availability of specific elements called nutrients are essential to achieve high rates of biodegradation and destruction of the target hazardous organic compounds. Macronutrients and micronutrients are elements that are respectively required in moderate and trace quantities. The required macronutrients in soils have been identified as the elements carbon, hydrogen, oxygen, nitrogen, phosphorus, potassium, calcium, magnesium and sulfur. A typical concentration for a macronutrient would be 50 ppm. A listing for required micronutrients or trace elements required in quantities smaller than 50 ppm are iron, manganese, zinc, copper, boron, molybdenum, cobalt, chlorine, and occasionally, vitamins, silicon, iodine, fluorine, vanadium, and sodium.
Experience with soil biodegradations typically confirm that natural soil will contain all nutrients except for occasional shortages of the macronutrient nitrogen, and sometimes phosphorus or sulfur. Table 4 shows the oxygen uptake by a microbial population biodegrading a production pit sludge. The oxygen uptake by the microbial population is a measure of the metabolic rate of destruction of the organic material present. An additional 50 ppm of the three macronutrients nitrogen, sulfur, and phosphorus were added individually to each of three respirometers (one nutrient to each respirometer). Table 4 shows there was a significant increase in the oxygen uptake in the respirometer that had soluble nitrogen added. The biodegradation rate in that instance increased approximately fivefold. The macronutrient that may be in short supply at a field site is typically nitrogen.